The ubiquitin (Ub) proteasome system is important for maintaining protein homeostasis and has various roles in cell signaling, proliferation, and cell cycle regulation. In mammals, Ub is encoded by two monoubiquitin and two polyubiquitin genes. Although reduced levels of Ub due to the disruption of one polyubiquitin gene are known to decrease cell proliferation, the effect of disrupting both polyubiquitin genes remains elusive. Polyubiquitin gene Ubc knockout mice are embryonically lethal and polyubiquitin gene Ubb knockout mice are infertile. Thus, it is difficult to study the effects of double knockouts (DKOs). In the present study, the CRISPR/Cas9 system was used to simultaneously knockout both polyubiquitin genes, UBB and UBC, in HEK293T and HeLa cells. In DKO cells, growth decreased significantly compared to the control cells. We observed reduced proteasome function and reduced levels of free Ub in DKO cells. However, the levels of purified proteasome were not different between control and DKO cells, although the mRNA levels of proteasomal subunits were significantly increased in latter. We propose that the reduction of Ub levels, by disruption of both polyubiquitin genes, resulted in an altered proteasomal status, leading to the reduced proteasome activity, and decreased cellular proliferation.

中文翻译：

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同时破坏两种多聚泛素基因会影响蛋白酶体功能并减少细胞增殖。

泛素 (Ub) 蛋白酶体系统对于维持蛋白质体内平衡很重要，并且在细胞信号传导、增殖和细胞周期调节中具有多种作用。在哺乳动物中，Ub 由两个单泛素和两个多聚泛素基因编码。尽管已知由于破坏一个多聚泛素基因而导致的 Ub 水平降低会降低细胞增殖，但破坏两种多聚泛素基因的效果仍然难以捉摸。多聚泛素基因Ubc敲除小鼠胚胎致死，多聚泛素基因Ubb敲除小鼠不育。因此，很难研究双敲除 (DKO) 的影响。在本研究中，CRISPR/Cas9 系统用于同时敲除多聚泛素基因UBB和UBC, 在 HEK293T 和 HeLa 细胞中。在 DKO 细胞中，与对照细胞相比，生长显着下降。我们观察到 DKO 细胞中蛋白酶体功能降低和游离 Ub 水平降低。然而，纯化的蛋白酶体水平在对照和 DKO 细胞之间没有差异，尽管后者的蛋白酶体亚基的 mRNA 水平显着增加。我们提出，通过破坏两个多聚泛素基因来降低 Ub 水平会导致蛋白酶体状态改变，从而导致蛋白酶体活性降低和细胞增殖减少。